DNA detector and DNA detection method

ABSTRACT

An electrophoresis apparatus includes a plurality of first capillaries containing a separation medium, such as a gel, and an optical cell in which one end of each of the first capillaries is disposed. Samples labeled with fluorophores are introduced into the first capillaries, and an electric field is applied to the first capillaries to cause the samples to migrate through the first capillaries into the optical cell. A light source excites the fluorophores with light when the samples are in the optical cell, causing the fluorophores to emit fluorescence, and a photodetecting system detects the fluorescence emitted by the fluorophores. The ends of the first capillaries in the optical cell may be arranged in a straight line. The light source may include a He-Ne laser emitting light having a wavelength of 594 nm, and the fluorophores may include either sulforhodamine or a derivative of sulforhodamine. The apparatus may include a plurality of second capillaries each having one end disposed in the optical cell such that the ends of the second capillaries are separated from the ends of the first capillaries by respective gaps, or may include a single second capillary having one end disposed in the optical cell such that the end of the single second capillary is separated from the ends of the first capillaries. A sheath solution may be introduced into the optical cell to form sheath flows of the sheath solution around the ends of the first capillaries.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 051,324 filedon Apr. 23, 1993, now abandoned, which is a continuation-in-part ofapplication Ser. No. 026,592 filed on Mar. 5, 1993, now U.S. Pat. No.5,314,602, which is a continuation of application Ser. No. 843,232 filedon Feb. 28, 1992, now U.S. Pat. No. 5,268,080, which corresponds toJapanese application No. 3-34006. The disclosures of application Ser.Nos. 026,592 and 843,232 are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a method of detection of DNA andprotein and of DNA base sequencing determination and to an apparatustherefor.

It relates more particularly to the fluorescence detection type gelelectrophoresis apparatus.

For DNA base sequencing determination by electrophoresis gel separation,a radioisotope label has been used as a label for a DNA fragment. Due tothe inconvenience of this method, however, a method of using afluorescence label has come to be increasingly employed. (Refer to U.S.patent application Ser. No. 07/506,986 (U.S. Pat. No. 5,062,942) andBio/Technology Vol 6, July 1988, pp816-821, for example.) As anexcitation light source this method uses an argon laser with an outputof 20 to 50 mw and a wavelength of 488 nm or 515 nm to detect the DNAfragment of 10⁻¹⁶ mole/band to 2×10⁻¹⁸ mole/band. As fluorophores, themethod has used FITC (fluorescein isothiocyanate with a maximum emissionwavelength of 515 nm), SF (succinyl fluorescein with a maximum emissionwavelength of 510 nm to 540 nm), TRITC (tetrarhodamine isothiocyanatewith a maximum emission wavelength of 580 nm) and Texas Red(sulforhodamine 101: a maximum emission wavelength of 615 nm).

Normally, electrophoresis is performed with polyacrylamide gel placed onthe plate which is provided between two glass plates. In recent yearsthe capillary gel electrophoresis method has been developed, where gelis formed in the capillary. Use of a capillary having a smaller diameterincreases the surface area per volume of gel; this feature facilitiesthe dissipation of Joule heat, permitting application of high voltage. Ahigh-speed electrophoresis separation is provided by the capillary gelelectrophoresis method.

The first example of the capillary gel electrophoresis method as a priorart is described in Nucleic Acid Research, Vol. 18. pp. 1415 to 1419(1990), Journal of Chromatography, Vol. 516, pp. 61-67 (1990), andScience, Vol. 242, pp. 562-564 (1988). Another method of using onemigration lane for base sequence determination is disclosed in NatureVol. 321, pp. 674-679 (1986) and others.

The above-mentioned conventional technique, however, has disadvantagesin that the sensitivity is insufficient, and the entire equipment mustbe made greater in size because the Ar laser is greater in size than aHe-Ne laser.

SUMMARY OF INVENTION

The first object of the present invention is to provide a solution tothe above-said problems and to provide a method and small-sized devicein which extra-sensitive DNA detection is made possible. To achieve theobject, the present invention uses a He-Ne laser with an emissionwavelength of 594 nm in DNA base sequencing determination byfluorescence detection type electrophoresis gel separation, and adopts ahighly efficient photodetecting system.

The said examples of the prior art use one capillary, but sufficientconsideration has not been given to simultaneous processing of two ormore samples. In the capillary electrophoresis apparatus, the decreasingsize of the samples requires higher detection sensitivity andsimultaneous processing of two or more samples. For fluorescencedetection in the state of electrophoresis, generally, the backgroundfrom the gel, scattered light from the inner and outer walls of thecapillary as the gel support or fluorescence from the capillary itselfis produced in addition to the fluorescence from the object fluorophoreitself, resulting in higher background level and reduced detectionsensitivity. One of the major problems in ensuring highly sensitivefluorescence detection is how to cut off such backgrounds. Improvementof processing capabilities and simultaneous processing of two or moresamples require an increase in migration speed, or detection bymigration of the DNA fragments through simultaneous use of two or morecapillaries which are migration lanes. In practice, there remains aproblem of how to cut off the background to achieve highly sensitivefluorescence detection, as mentioned above.

The second object of the present invention is to solve said problems toprovide a capillary electrophoresis apparatus and its method whichensure fluorescence detection of two or more samples, and high-speedhighly sensitive DNA detection.

The measuring limit for the DNA fragment labeled by a fluorophore in theprocess of electrophoresis gel migration is determined by the intensityand fluctuation of the background fluorescence from the gel with respectto fluorescence for labeling. The background fluorescence from the gelis gradually reduced with the increase of the emission wavelength.

Thus, the excited at the optimum wavelength, the quantity of thefluorescence normalized by the background fluorescence from the gel isthe greatest in the case of Texas Red (sulforhodamine 101). According tothis normalized quantity of the fluorescence, the sensitivity of theTexas Red is five to ten times that of FITC. In the present invention,there has been used Texas Red or its derivative as a labelingfluorophore, and a He-Ne laser with the wavelength of 594 nm, which isclose to the optimum wavelength, as the excitation light source. Thewavelength of 594 nm for the excitation light is close to the maximumemission wavelength of the Texas Red which is 615 nm. One of theproblems was how to remove the scattered light from the excitationlight, and the present invention has succeeded in removing thisscattered light by using a sharp-culling fluorophore filler which willbe described below. The output from the He-Ne laser with the wavelengthof 594 nm ranges from 1 mw to 7 mw. The output from a typical model ofthe He-Ne laser (594 nm) is as small as 2 mw, and greater emissionstrength cannot be obtained; therefore, the detecting sensitivitygreatly depends on the fluctuation of the background fluorescence fromthe gel. To solve this problem, the present invention has improved thephotodetecting system, and has adopted a photodetecting system whichreceives a greater amount of light by two or more digits than theexcitation light scanning method. Namely, the excitation light is madeto be incident upon the gel plate through the side thereof, and theentire measured area is irradiated simultaneously to increase theoverall emission strength. Furthermore, a cylindrical lens is used toincrease the photodetecting solid angle.

Changing the wavelength of the excitation light from 488 nm to 594 nmhas reduced the background fluorescence from the gel down toapproximately one fifth when the laser of the same output is used. Inaddition, when the conventional argon laser (about 20 mw) is employed,FITC is subjected to photodestruction, and this results in reducedemission strength, and hence reduced sensitivity. By contrast, under the2.5 mw He-Ne laser irradiation, photodestruction of the fluorophorehardly occurs to the Texas Red during measurement. This permits theemission strength normalized by the background fluorescence from the gelto be greater by one digit than that of FITC.

In the laser scanning method, the area of 100 mm is swept by the laserbeam of approximately 0.3 mm in diameter. Even when the conventional 50mw laser is used, the average laser intensity with which each point isirradiated is as small as 17 microwatts, since the irradiation time ateach point is reduced. When the 2.5 mw laser is used, the average laserintensity is approximately 0.9 microwatts, and this almost cannot be putinto practical use. The irradiation intensity is 2.5 mw in the lateralincidence method employed in one of the present embodiments, and thisemission is sufficient. However, in the scanning method thephotodetecting efficiency can be made approximately 2 percent, but inthe simultaneous irradiation method, the fluorescent image is receivedin a reduced size; therefore the photodetecting efficiency is reduced to0.1 percent or less.

Defining a solid angle as Ω and the transmittance of the filter or thelike as T, the photodetecting efficiency η can be expressed by thefollowing formula (1): ##EQU1##

Assuming the image reduction ratio as m and the f-number of the lens asF, Ω is represented as: ##EQU2##

Thus, the photodetecting efficiency η can be expressed by the followingformula (3): ##EQU3## where m represents (length of the measuredportion)/(length of the detector). In the scanning method, m<1; and inthe lateral incident method, 120 cm/24 cm<m<120 cm/18 cm by way of anexample, namely m is approximately from 5 to 7. Accordingly, thephotodetecting rate of the lateral incident method is approximately1/50, because of the term of (m+1)² in formula (3), on the one hand. Onthe other hand, in the case of the scanning method where light is notcontinuously received from each measured point, the result is multipliedby 3/1000 to 5/1000 as a factor due to duty cycle. Thus, in total, thelateral incident method yields a greater photodetecting efficiency thanthe scanning method. When the laser having a smaller output such as aHe-Ne laser is used, it is important to find a means to obtain asufficient photodetecting efficiency. The present invention uses thecylindrical lens to increase the photodetecting efficiency by, forexample, four to five times. This system provides a high photodetectingefficiency, ensuring highly sensitive detection of the fluorescentimage.

U.S. patent application Ser. No. 07/506,986 discloses the case of usingTexas Red and the He-Ne laser having a wavelength of 543 nm. Comparedwith the case of using the 594 nm He-Ne laser, the excitation efficiencyis as low as 1/3, and the output is also as low as 1 mw.

To achieve the second object, in the electrophoresis apparatus whereinsamples DNA fragments, etc. labeled by the fluorophore are subjected toseparation by electrophoresis, providing optical detection and analysisof the separated samples, an optical detecting portion for sampledetection, for which the electrophoresis separation region providedbetween the vessel for cathode electrode and vessel for anode electrodeis composed of capillaries, has the following configurations:

In the configuration (1) of said optical detecting portion; the ends ofsaid one pair or more pairs of capillaries are connected to said vesselfor cathode or anode electrode, and the other ends are held at aspecified gap, with their axes almost matched to each other, and arelaid face to face with each other in the optical cell to form amigration lane which passes through said optical cell; sheath solutionis supplied into said optical cell from the outside; the sample migratedfrom the capillary end of the upstream migration lane, namely, thesample separation region, is put in the sheath flow condition, and isthen lead into the downstream capillaries laid out face to face; whilesaid gap is used as an optical detecting portion, and light from thelight source is shed on this optical detecting portion, therebydetecting the samples. In this configuration (1), two or more detectingportions formed by two or more pairs of capillaries are laid out in theoptical cell. Furthermore, two or more pairs of capillaries are arrangedin the optical cell so that two or more detectors formed by two or morepairs of capillaries are located in a straight line, and the excitationlight is shed along said straight line so that all the optical detectingportions are simultaneously irradiated, thereby ensuring simultaneousdetection of the fluorescence at said two or more optical detectingportions.

In the configuration (2) of the optical detecting portion; two or morecapillaries, the other ends of which are immersed in the electrodevessel, are terminated in the optical cell, and sheath solution issupplied into said optical cell from the outside. Thus, the samplesmigrating from capillaries are made to flow in the optical cell in thesheath flow condition. Using the sheath flow region as the opticaldetecting portion, light is irradiated on the optical detecting portion,thereby detecting the samples. In this configuration (2), two or morecapillaries are laid out in the optical cell so that two or more opticaldetecting portions are located in a straight line, and the excitationlight is shed along said straight line so that all the optical detectingportions are simultaneously irradiated, thereby ensuring simultaneousdetection of the fluorescences issued from the samples migrating fromcapillaries.

In configurations (1) and (2), the following configuration is alsopossible:

The sample separation region is composed of the capillary gel, and thesheath solution has the same components as those of the buffer solutioninside the capillaries. The denaturant for the sample may be containedas required. The sheath solution level is positioned higher than theliquid level in the downstream electrode vessel, and the sheath solutionis made to flow by the head of two liquids.

In the configuration (3) of the optical detecting portion; two or morecapillaries, the other ends of which are immersed in the electrodevessel, are terminated in the optical cell filled with electrolyte. Theoptical detecting portion belongs to the region close to the terminal towhich the samples migrate from the capillary gel as migration lane. Theoptical detecting portion filled with electrolyte is formed as follows:two capillaries are laid out in a straight line with their axes almostmatched to each other, and the ends laid face to face with each otherare placed in close contact with each other in the axial direction ofthe capillary, while maintaining a specified gap. In this case, samplesare subjected to electrophoresis separation inside one of thecapillaries located in the upstream side of the migration lane, whilethe gap serves as optical detecting portion to detect the fluorescenceemitted by samples. The gap length is preferred to be 1 mm or less. Thetwo or more optical detecting portions formed by two or more pairs ofcapillaries are linked with each other by the electrolyte. Two or moreoptical detecting portions are arranged in a straight line, and a singleexcitation light is shed along this straight line, ensuring simultaneousdetection of the fluorescences issued from the two or more samples.

In the electrophoresis apparatus provided with optical detecting portionaccording to said configuration (1), sheath solution is supplied intothe optical cell from the outside, so samples migrating from thecapillary end in the migration lane on the upstream side where thesamples are separated can be led to the capillaries facing each other inthe sheath flow condition, and samples migrate continuously incapillaries on the upstream side and those on the downstream side.Furthermore, the samples migrate smoothly in the gap on the axis betweenthe capillaries on the upstream side and those on the downstream sides.This gap is used as the optical detecting portion. Light can beirradiated on the optical detecting portion in the sheath solutioncontaining no capillaries, detecting the samples by fluorescence. Thiseliminates the possibility of backgrounds being emitted from capillariesor capillary gels, ensuring highly sensitive fluorescence detection. Itallows use of the rectangular optical cell which can be manufacturedeasily at lower cost. Two or more optical detectors can be arranged inclose proximity with each other in the optical cell, resulting insubstantial reduction of the system size. Samples migrating from thecapillaries are put into sheath flow condition for each capillary whenpassing through the optical detecting portion, and sheath flow is allput under the same conditions. Sheath flow conditions such as flow speedare made uniform for each capillary, resulting in improved accuracy inoptical detection of samples. Two or more optical detecting portions arearranged in a straight line in one optical cell, and excitation light isirradiated along this straight line, permitting simultaneous irradiationof all optical detecting portions and simultaneous fluorescencedetection by two or more optical detecting portions. Furthermore, two ormore optical detecting portions are linked with each other through thesolution, so the excitation light is not bent by capillaries, and theexcitation light intensity is not damped. This allows irradiation of theoptical detecting portions with sufficient light intensity, providinghigh-precision highly sensitive fluorescence detection. Use of thetwo-dimensional TV camera, etc. for light detector permits simultaneousphotodetection of the fluorescent images of two or more opticaldetecting portions. Two or more optical detecting portions can bepositioned in close proximity with each other in a straight line, andthe length between the optical detecting portions on the extreme ends ofthis straight line can be reduced, permitting configuration of thesmaller apparatus. Reduced distance between optical detecting portionslocated on the extreme ends will allow all optical detecting portions tobe irradiated in almost the same light beam diameter, even when theexcitation light is condensed by the lens or the like.

For example, when the laser light is condensed to 100 μm in terms of thefocal point, the laser light diameter will be about 100 μm over therange of about 10 mm on the front and rear of the focal point. Whencapillaries having an outer diameter of 200 μm and inner diameter of 100μm are arranged at the intervals of 400 μm, about 50 capillaries can beinstalled within the range of about 10 mm on the front and rear of thefocal point, and they can be irradiated with the equivalent light beamdiameter and intensity. This allows the optical detecting portions to beirradiated with the excitation light condensed, and the fluorescenceintensity to be increased, thereby ensuring highly sensitive detectionof the sample.

Moreover, it is also possible to make the downstream capillaries hollow(i.e. open capillaries), allowing effective flowing of the sheathsolution. In addition to the capillaries, it is also possible to use onthe downstream side something that performs the equivalent operations,for example, the plate provided with holes and grooves in the samenumber as that of the upstream capillaries. The flow of electricity atthe gap (namely, the optical detecting portion) can be ensured by makingthe sheath solution have the equivalent components as that of the buffersolution within the capillary, thereby providing electrophoresis ofsamples. Furthermore, because the buffer solution has the samecomponents both inside and outside the capillary, there is nopossibility of the buffer solution inside the capillary flowing out intothe optical cell causing their composition to be changed. Therefore, thesample separation function in electrophoresis is not lost. When thesamples are single-strand DNAs, denaturant can be contained in thesheath solution, and it is possible to avoid rebonding when DNA samplesmigrates in the gap (namely, the optical detecting portion); this meansimproved detection accuracy. This feature reduces the possibility of thedenaturant contained in the capillary leaking out into the optical cell,and eliminates the loss of sample separation function.

The gap length is preferred to be 0.1 mm to 3.0 mm. Generally, thesmaller gap length provides easier electrophoresis of the samples in thegap space, so the space distance should be short. However, assembling ofthe apparatus is more difficult if the gap length is very small;therefore, it is preferred to be 0.1 mm or more in practice. However, itcan be set to 0.1 mm or less. The limit is determined by the size of theexcitation beam such as laser light at the gap. Conversely, greater gaplength will cause easier dispersion of the samples. The gap length ofabout 3.0 mm allows normal electrophoresis of the samples on the lineconnecting between capillaries.

In the electrophoresis apparatus provided with optical detecting portionaccording to said configuration (2), samples migrating in thecapillaries flow in the optical cell in the sheath flow state. By usingthe sheath flow region as optical detecting portion and the sameconfiguration as that of (1), it is also possible to detect separatelythe samples migrating in the capillaries, thereby obtaining the sameresults as configuration (1). The layout of the optical detectingportion and irradiation of the excitation light discussed in connectionwith the configuration (1) are the same for configuration (2).

In the configuration (3) of the electrophoresis apparatus; samples aredetected in the electrolyte without containing any gel, eliminating thepossibility of backgrounds being emitted from gel supports such as thecapillaries, ensuring highly sensitive fluorescence detection. Moreover,it eliminates the need of making the electrolyte, and provides simpleconfiguration of the apparatus. The gap is used as optical detectingportion to detect the fluorescence of samples; it is also filled withelectrolyte, permitting electrophoresis. Since gel is produced in atleast one of the capillaries, formation of the migration lane isfacilitated. The preferred gap length is 0.1 mm to 1 mm. Generally, thesmaller gap length provides easier electrophoresis of the samples in thegap space, so the space distance should be short. However, assembling ofthe apparatus is more difficult if the gap length is very small;therefore, it is preferred to be 0.1 mm or more in practice. However, itcan be set to 0.1 mm or less. The limit is determined by the size of theexcitation beam such as laser light at the gap.

Conversely, greater gap length will cause easier dispersion of thesamples without migrating in a straight line in the gap; samples willnot migrate in the others of the paired capillaries. The gap length ofabout 2 to 3 mm allows normal electrophoresis of the samples; however,normal electrophoresis may take place, depending on the conditions suchas electrophoresis voltage. The gap length of about 1 mm is preferred inpractice. That is, the gap length of 0.1 mm to 1.0 mm allows smooth andeffective electrophoresis of the samples. Two or more optical detectingportions can be positioned in a straight line, and a single excitationlight can be irradiated simultaneously on two or more optical detectingportions for fluorescence detection. Furthermore, two or more opticaldetecting portions are linked with each other through the solution, sothe excitation light is not damped. This allows irradiation of theoptical detecting portions with sufficient light intensity, providinghigh-precision highly sensitive fluorescence detection.

To summarize the present invention, one or more samples are subjected toelectrophoresis separation using the plate gel and capillary gel, andtwo or more migration lanes are irradiated linearly by the laser fromthe direction which is almost perpendicular to the migration directionfor samples and which is parallel to the surface formed by two or moremigration lanes, thereby providing real-time detection of thefluorescence emitted from the fragments migrating in the migration lane.The present invention relates especially to the fluorescence detectiontype electrophoresis apparatus provided with the optical cell which isintended for highly sensitive fluorescence detection of the DNAfragments labeled by fluorophores, wherein one pair or more pairs ofcapillary filled with gel and capillary are arranged in the optical cellso that they are coaxial with each other and a specified gap length ismaintained. The sheath solution is poured into the optical head by thecell and the samples migrating in the gap are put in the sheath flowcondition. Fluorescence detection is performed in the gap free ofcapillary or gel. Or the buffer solution is poured in the optical cell,and the gap is filled with buffer solution, thereby forming themigration lane through the gap, which is used for fluorescencedetection. Using sulforhodamine 101 or rhodamine derivative asfluorophore, He-Ne laser light having an emission wavelength of 594 nmis irradiated along the straight line in which two or more gaps arearranged; then the fluorophore is excited to permit fluorescencedetection. Since the fluorescence detection is performed in the gap freeof capillary or gel, it is possible to obtain a small typeelectrophoresis apparatus which provides simultaneous electrophoresis oftwo or more samples and their simultaneous detection, thereby ensuringhighly sensitive fluorescence detection, free from background influence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view representing the DNA detector as the firstembodiment of the present invention;

FIG. 2 is a graph representing the spectrum in the electrophoresisseparation of the DNA fragment which is obtained by letting the λ phagebe digested by the restriction enzyme and by inserting the fluorescencelabel into the cut portion;

FIG. 3 is a block diagram representing the electrophoresis region andlaser irradiation system of the electrophoresis apparatus according tothe second Embodiment of the present invention;

FIG. 4 is a block diagram representing the fluorescence detection systemof the electrophoresis apparatus according to the second Embodiment ofthe present invention;

FIG. 5 is a block diagram representing the electrophoresis region andlaser irradiation system of the electrophoresis apparatus according tothe fourth Embodiment of the present invention;

FIG. 6 is a block diagram representing the electrophoresis region andlaser irradiation system of the electrophoresis apparatus according tothe fifth Embodiment of the present invention;

FIG. 7 is an oblique view representing the plate provided with two ormore grooves according to the sixth Embodiment of the present invention;

FIG. 8 shows the optical cell of the electrophoresis region of theelectrophoresis apparatus according to the sixth Embodiment of thepresent invention;

FIG. 9 is a block diagram representing the electrophoresis apparatusaccording to the seventh Embodiment of the present invention;

FIG. 10 is an enlarged view representing the optical cell according tothe seventh Embodiment of the present invention;

FIG. 11 is a block diagram representing the electrophoresis apparatusaccording to the eighth Embodiment of the present invention; and

FIG. 12 is an enlarged view representing the optical cell according tothe ninth Embodiment of the present invention;

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following gives detailed description of the present invention withreference to embodiments:

EMBODIMENT 1

First embodiment of the present invention will be described withreference to FIGS. 1 and 2. FIG. 1 is a schematic view representing thedetector. Light emitted from the 594 nm He-Ne laser 1 irradiates theelectrophoresis separation gel plate 4 from the side. After beingcollected by the cylindrical lens 7, the fluorescence emitted from thelinearly irradiated portion is filtered out by the band pass filter 6,and forms images on the line sensor or secondary detector 8 through thelens for image formation 5. Filler 6 is a 6-cavity multilayerinterference filter with a diameter of 50 mm, and transmittances forlight having wavelengths of 594 nm, 600 nm, 610-630 nm and 640 nm are10⁻⁴, 10⁻², 0.6 or more, and 0.01 or less, respectively.

The concentration of the acrylamide gel constituting the gel plate 4(concentration of the total quantity of monomer) is 4 to 6 percent(g/cc). When irradiated by the He-Ne laser with the wavelength of 594nm, the background fluorescence from the gel has the same intensity asthe fluorescence from Texas Red having a concentration of 2×10⁻¹¹ mole.The laser power is 2.5 mw and the beam diameter is 0.3 mm. Thepositional resolution on the linearly irradiated portion of 0.5 mm issufficient. In FIG. 1, the reference numeral 2 denotes a reflectionmirror, 3 a glass plate sandwiching the gel plate 4 sandwiched, 9 acontrol circuit, and 10 a data processor. The number of the photons I ofthe fluorescence emitted from the 0.5 mm-long area irradiated by thelaser beam can be obtained from the following formula:

    I˜I.sub.0 ·φ·[l-e.sup.- lM ]˜I.sub.0 O lM(4)

where I₀ denotes the number of incident photons, O denotes a quantumyield of the fluorophore, denotes a molar absorption coefficient, ldenotes a optical path length and M denotes a mole concentration of thefluorophore. The number of the photons emitted from the 2.5 mw laser persecond (namely, I₀) is approximately 10¹⁶. When Texas Red ofapproximately 0.4 in O is irradiated with the light having thewavelength of 594 nm, the absorption coefficient of Texas Red is approx.8×10⁴ cm⁻¹ (M)⁻¹, l is approx. 0.05 cm, and the concentration M of TexasRed showing the same level of fluorescence as that of the gel is approx.2×10⁻¹¹ moles/l. The number of photons I from Texas Red which emits thesame amount of the fluorescence as that of background fluorescence fromthe gel is estimated to be approximately 3×10⁸ per second.

When a 10 cm area is scanned by the laser beam, the duty cycle is0.5/100. Therefore, the average number of photons emitted from the 0.5mm-long area is 1.5×10⁶ per second. Even when the lens having a greaterF-value is used to receive light, the photodetecting efficiency is 1 to2 percent when consideration is given to the filler transmittance(approximately 50%). When consideration is given to the quantum yield(approximately 5%) on the photodetecting surface, the number of photonsto be received is 1000 per second or less. Thus, the scanning methodfails to provide high-precision measurement.

In the lateral incident method proposed in the present invention,however, the duly cycle is 1.0, but since the reduced image is formed onthe detector, the photodetecting efficiency is as small as approx.0.05%. The number of protons emitted from the 0.5 mm-long area whichenter the detector is approx. 7.5×10³ per second when the quantumefficiency on the photodetecting surface and losses due to variousfactors are taken into consideration. The detection sensitivity isdetermined by the fluctuation of the background fluorescence to bemeasured.

In this case, the statistical fluctuation is approximately ±1.2 percent.Generally, the relative value of the fluctuation is reduced with theincrease of the photodetecting quantity, and even a slight signal can bemeasured. If the photodetecting quantity is increased by N times, therelative fluctuation is reduced to ±1/√N. For example, when thephotodetecting quantity is increased by four times, the relativefluctuation is reduced by one half. To ensure highly sensitivedetection, the above-said fluctuation of approx. ±1.2% must be furtherreduced, and the photodetecting quantity must be increased.

To realize this, the present invention uses a cylindrical convex lens(focal distance f=25 mm, f-number F=1.0) which is installed at aposition approximately 25 mm away from the irradiation section, and acylindrical concave lens (f=-200 mm) which is placed immediately beforethe lens for image formation so that the image in the vertical directionwill be formed in an enlarged size; hence the photodetecting solid anglehas been increased four to five times. This has increased thephotodetecting quantity by four to five times, and has reducedfluctuation by half down to approximately ±0.6% of the fluorescenceemitted from the gel, thereby ensuring a highly sensitive detectioncapability. Namely, this detection system permits detection of Texas Redof 2×10⁻¹³ moles/l at an S/N ratio of approximately 1.

When the argon laser is used as an excitation light source, and TexasRed is used as a labeling fluorophore, the excitation efficiency isreduced by one digit compared with that of the present embodiment, andthe sensitivity is also reduced undesirably by one digit. When the argonlaser is used as an excitation light source, and FITC is used as alabeling fluorophore, the background fluorescence is increased by onedigit compared with that of the present embodiment, and the labelingfluorophore is subjected to photodestruction during the measurement sothat the effective FITC concentration is reduced. As a result, thesensitivity is also reduced undesirably by two digits compared with thatof the present embodiment.

FIG. 2 represents an electrophoresis separation pattern of the fragmentwhich is digested by the enzyme wherein the terminal of the λ phage islabeled by Texas Red and the He-Ne laser of 594 nm wavelength isemployed. The cubic volume of the DNA band is estimated at 1 μl, and itis possible to read the signal from the sample which is injected 2×10⁻¹⁹moles.

By contrast, the quantity of the sample for which the signal can be readis 1×10⁻¹⁷ moles per band in the conventional case of using FITC and theargon laser, and is 2×10⁻¹⁸ moles per band in the conventional case ofusing Texas Red and the argon laser.

The following Table shows the comparison between an example of the He-Nelaser used in the present invention and an example of the argon laserused in the conventional case. This reveals that the He-Ne laser issmaller in size, lighter in weight and usually less costly than theargon laser.

    ______________________________________                                               He-Ne-laser   Argon laser                                                              Weight              Weight                                           Size (cm)                                                                              (kg)     Size (cm)  (kg)                                      ______________________________________                                        Power supply                                                                           8 × 15 × 15                                                                  2        15 × 40 × 30                                                               20                                      Resonator                                                                              7 (dia.) × 4                                                                       2        15 × 15 × 35                                                               10                                      ______________________________________                                    

Thus, the DNA detector of the present invention features not only ahigher sensitivity but also a smaller size than the conventional device.

As described above, according to the present invention, Texas Red orrhodamine derivatives having an emission band in the long wave area ofless background fluorescence from the gel can be effectively excited bythe yellow He-Ne laser with the wavelength of 594 nm, so that thischaracteristic ensures a higher sensitivity and a smaller configuration.

EMBODIMENT 2

In the present Embodiment, DNA fragments labeled by fluorescence areseparated by electrophoresis and detection is made by fluorescence. Thefollowing describes the case of using Texas Red (Sulforhodamine 101,maximum emission wavelength of 615 nm) as labeling fluorophore. The DNAfragments as samples are labeled by fluophores. DNA fragments labeled byfluorophores are prepared by DNA polymerase reaction, using the primerlabeled by fluorophore according to the well-known dideoxy sequencingmethod invented by Sanger and his colleagues. The details of the methodof preparing samples according to Sanger's dideoxy sequencing method aredescribed in the Embodiment 3.

Firstly, the apparatus configuration will be described. FIG. 3 shows theelectrophoresis region and laser irradiation system of theelectrophoresis apparatus according to the present Embodiment. FIG. 4represents the fluorescence detection system of the electrophoresisapparatus. The electrophoresis apparatus includes an arrangement of 20capillaries serving as the electrophoresis region to performsimultaneous detection of two or more samples. The electrophoresisseparation region uses 20 silica-made capillaries 1a, 1b, 1c, 1d . . .1t, all having the same form; an inner diameter of 100 μm, outerdiameter of 375 μm and length of 40 cm, as well as 20 silica-madecapillaries 2a, 2b, 2c, 2d . . . 2t, having the same inner and outerdiameters but a length of 10 cm.

The capillary gel is produced by filling capillaries 1a to 1t withpolyacrylamide gel containing the denaturant urea. Firstly, thecapillary interior is washed and is subjected to silane couplingtreatment. Then solution of N,N,N',N'-tetramethylethylendiamine andammonium persulfate is added to the degassed TRIS-borate buffercontaining 3.84 percent of acrylamide, 0.16 percent of N,N'-methylenebis-acrylamide, 7M of urea, and 2 mM of EDTA, and is poured into thecapillaries; then polyacrylamide gel is obtained by polymerization.Polyacrylamide gel and capillaries are chemically bonded with eachother, and the gel does not come out of the capillaries duringelectrophoresis.

Capillaries 2a to 2t are treated so that their inner surfaces arepositively charged. Firstly, 3-(2-aminoethylaminopropyl) trimethoxysilansolution is poured into capillaries to cause reaction. It is then heattreated at the temperature of 110° C., and amino acid residue isintroduced on the inner walls of the capillaries to be positivelycharged. This changes the direction of the electroosmotic flow fromnegative to positive poles inside each of capillaries 2a to 2t.

The migration direction (from negative to positive poles) of samples incapillaries 1a to 1t filled with polyacrylamide gel is matched to themigration direction (from negative to positive poles) of samples incapillaries 2a to 2t, ensuring the sample migration. The ends ofcapillaries 1a to 1t and capillaries 2a to 2t are placed face to face inthe optical cell, and are held at a specified gap; then samplesmigrating in these gaps are detected optically. According to the presentinvention, the fluorescent cell is used to detect the samples byfluorescence. That is, the ends of said capillaries 1a to 1t andcapillaries 2a to 2t are placed inside the rectangular quartz opticalcell 104a (outer dimensions: 36 mm wide by 4.5 mm deep by 3 mm long;inner dimensions: 30 mm wide by 2 mm deep by 3 mm long); where widthdenotes the horizontal direction of the drawing (direction ofcapillaries 1a→1t), the depth the perpendicular direction of thedrawing, and the length the longitudinal direction (direction of samplesmigrating in capillaries) of the drawing. They are arranged so that apair of capillary 1a and capillary 2a will be coaxial with each other,and that they will face each other forming the gap 3a having a length of1 mm. Likewise, capillaries 1b and 2b, 1c and 2c, 1d and 2d . . . 1t and2t are arranged so as to form gaps 3b, 3c, 3d . . . 3t. Gaps 3b, 3c, 3d. . . , 3t are arranged in a straight line at a specified interval. Tohold the capillaries in the optical cell, use is generally made of themulti-capillary holder having 20 vertical holes at intervals of 0.6 mmprovided on the plate-formed block made of fluorine-contained polymer,for example, tetrafluoroethylene polymer. Namely, each of capillaries 1ato 1t is inserted in each of 20 vertical holes of multi-capillary holder5a; then each of capillaries 2a to 2t is inserted in each of 20 verticalholes of multi-capillary holder 5b. The multi-capillary holder 5a andmulti-capillary holder 5b are fixed in close contact with the top andbottom of optical cell 104a to ensure that capillaries 1a and 2a, 1b and2b, 1c and 2c, 1d and 2d . . . 1t and 2 are respectively coaxial.

Furthermore, since the gaps 3a to 3t are used as optical detectingportions, adjustment is made of the length of capillaries 1a to 1t and2a to 2t inside the optical cell 104a so that gaps 3a to 3t are laid outin a straight line. The other ends of the capillaries 1a to 1t and 2a to2t are immersed in a vessel for cathode electrode 106 and a vessel foranode electrode 107 supplied with buffer solution (TRIS-borate-EDTAbuffer solution with urea). The inside of optical cell 104a is providedwith sheath inlet 108 to be filled with sheath solution, so that sheathsolution 11 in the sheath solution bottle 100 can be supplied throughthe tetrafluoroethylene polymer tube 109. Sheath solution 11 uses theTRIS-borate-EDTA buffer solution with urea, having the same compositionas that of the buffer solution inside the capillary gel of capillaries1a to 1t, thereby preventing the components of the capillary gel fromleaking into the optical cell 104 a.

Furthermore, if optical cell 104a incorporating capillaries 1a to 1t andcapillaries 2a to 2t is filled with sheath solution and the level ofsheath solution 11 of sheath solution bottle 100 is made higher thanthat of the buffer solution in the vessel for anode electrode 107, thensheath solution flows into the vessel for anode electrode 107 throughcapillaries 2a to 2t. Under this condition, optical cell 104a andcapillaries 2a to 2t are filled with sheath solution, namely, buffersolution, and capillaries 1a to 1t are also filled with capillary gel.If the DC voltage is applied between the vessel for cathode electrode106 and the vessel for anode electrode 107 by DC high voltage powersupply 12, then current will flow through capillary 1a, gap 3a andcapillary 2a, allowing the samples to migrate. When the level of sheathsolution 11 of sheath solution bottle 100 is made higher than that ofthe buffer solution in the vessel for anode electrode 107, sheathsolution flows inside the capillaries 2a to 2t, producing the flow overthe tops of the capillaries 2a to 2t, namely, around gaps 3a to 3t.Samples migrating from capillaries 1a to 1t pass through gaps 3a to 3talong the flow over capillaries 2a to 2t under the sheath flowconditions, are led into each capillary and made to migrate toward thevessel for anode electrode 107.

Capillaries 2a to 2t are treated so that their inside will be positivelycharged, and electroosmotic flow inside the capillaries 2a to 2t isdirected from capillaries 1a to 1t vessel for anode electrode 107,eliminating the possibility of reserve flow of the solution into gaps,and ensuring stable flow of sheath solution toward the vessel for anodeelectrode 107. Even when the flow rate of sheath solution is especiallylow, stable flow of sheath solution toward the vessel for anodeelectrode 107 is ensured.

Electrophoresis is performed by application of DC power between thevessel for cathode electrode 106 and the vessel for anode electrode 107by means of DC high voltage power supply 12. The current flowing throughcapillary 1a and capillary 2a goes mainly through gap 3a; likewise, thecurrent flowing through capillaries 1b and 2b, 1c and 2c, 1d and 2d, . .. 1t and 2t goes mainly through 3b, 3c, 3d . . . 3t. Furthermore, asdiscussed above, the samples migrating from capillaries 1a to 1t flowalong the stream over capillaries 2a 2t, passing though the gaps 3a to3t under the sheath flow condition; then they are led into respectivecapillaries. Namely, samples migrating through the capillaries and gapsare made to migrate without contacting the samples migrating in theadjacent gaps. This permits fluorescence detection of the samplesmigrating in each gap, without being affected by the samples migratingin the neighboring gaps. In addition, the samples do not contact theinner surface of optical cell 104a; this feature eliminates theinfluence of the samples being absorbed to the optical cell 104a, andprovides spatial removal of the scattered light on the surface of theoptical cell 104a by means of slits or similar device. Thus, highlysensitive fluorescence detection is possible.

Introduction of the DNA fragments labeled by fluorophores is madepossible by immersing one end of the capillary 1a on the cathode sideinto the sample solution, and by application of the 6 kV-voltage betweenthe sample solution and the vessel for anode electrode 107 for about 20seconds. After that, the end of the capillary is put back to theoriginal position of the vessel for cathode electrode 106. Thisprocedure is repeated for each of capillaries 1a to 1t, to supplysamples into the capillary gels of capillaries 1a to 1t. Note thatsamples may be supplied to each capillary in sequence, as describedabove, or they may be supplied by immersing each of capillaries 1a to 1tinto the sample solution and by simultaneous application of voltage. Thesamples poured in each of the capillaries 1a to 1t are made to move fromthe cathode to anode by application of the 6 kV-voltage between thevessel for cathode electrode 106 and the vessel for anode electrode 107inside the capillary gels of the capillaries 1a to 1t, and pass throughgaps 3a to 3t. Samples passing through gaps 3a to 3t are dejected byirradiating He-Ne laser light having a wavelength of 594 nm to excitethe Texas Red (Sulforhodamine 101) which is a labeling fluorophore. Thelaser light is adjusted so that the laser light irradiates gaps 3a to 3tarranged in a straight line simultaneously or under much the sameconditions (laser light diameter), and the laser light is irradiated,thereby permitting fluorescence to be detected. Namely, laser light 21having a wavelength of 594 nm of the He-Ne laser source 20 is condensedby lens 22 and irradiated to excite the DNA fragments labeled byfluorophores passing through gaps 3a to 3t. The laser having a beamdiameter of about 0.7 mm, and the lens 22 having a focal distance of 100mm are used, and the focus is set to the mid-position between gaps 3aand 3t. In this case, spot size of the laser light at the focal point isabout 150 μm, and focal depth is about 20 mm. The distance between gap3a and 3t is equal to the distance from capillary 1a to capillary 1t. Inthe case of the present embodiment, it is 0.6 mm by 19, namely about 12mm. That is, the laser light irradiates 20 positions of the gaps 3a to3t with much the same spot size, which is much the same as that of thecapillary gel (100 μm). As discussed above, the spot size of the laserlight can be much the same as that of the capillary gel. Uniform andefficient excitation of the DNA fragments labeled by fluorophores whichmigrate through gaps 3a to 3t by selecting the light source and lenssystem so that all gaps are irradiated with much the same spot size.

The fluorescence emitted from the DNA fragments labeled by fluorophoreswhich migrate through gaps 3a to 3t is detected from the positionperpendicular to the direction of laser irradiation. This configurationis illustrated in FIG. 4. After the background such as scattered lighthas been eliminated through interference filter 32, fluorescence 30emitted from the DNA fragments, and forms the image on two-dimensionaldetector 34 such as CCD camera through lens 33. Being controlled bycontroller 35, two-dimensional detector 34 detects the fluorescent imageof gaps 3a to 3t, and provides continuous and simultaneous detection ofthe change of the fluorescence intensity according to all gaps 3a to 3t,using data processor 36 of the computer or the like. These results aredisplayed on monitor 37, and are output on printer 38 or stored inmemory 39. This feature permits simultaneous and continuous detection ofmigration patterns for each of capillaries 1a to 1t. Note that, in thecase of the present embodiment, the one-dimensional detector such as aphotodiode array can be used, instead of the two-dimensional detectorsuch as a CCD camera, since the fluorescent images of the gaps arearranged on the one-dimension basis. For effective detection of thefluorescence emitted from the Texas Red, interference filter 32 uses theband pass interference filter which permits transmission of a wavelengthband ranging from 610 to 630 nm.

The magnification of the lens is set so that the image of gaps 3a to 3twill be condensed on the photo-detecting surface of the two-dimensionaldetector. In the configuration shown in FIG. 4, fluorescence from thelinear laser irradiation region may be collected by the cylindricallens, as in the case of Embodiment 1, for which the fluorescencedetection system shown in FIG. 1. Since the gaps are filled with buffersolution in the present embodiment, the laser light irradiates gaps,without being affected by scattered light of the capillaries. Thispermits simultaneous, homogenous and efficient irradiation of samplesmigrating through two or more capillaries. It leads to substantialreduction of such background as scattered light and fluorescence fromcapillaries and capillary gels, ensuring highly sensitive fluorescencedetection. The effect of reducing the background light is described:Compared with the case where fluorescence detection is made by sheddingthe laser light on the capillary itself the coating of which is removed,for example, fluorescent detection made at the gap as in the presentembodiment reduces the detected background intensity to about one tenthor less, permitting detection of samples with smaller concentration.Arrangement of two or more gaps in a straight line enables simultaneousand simple irradiation of all gaps by the laser light, allowing simpleapparatus configuration. Since two or more pairs of capillaries can beheld in one optical cell, only one tube is sufficient for supply ofsheath solution. Sheath flow occurs only at the position close to thegaps, flow rates are the same for all gaps, resulting in greaterreproducibility. The optical cell according to the present embodimenthas a simple structure; it is not necessary to use the optical cell ofcomplicated configuration as found in the sheath flow chamber. In thepresent embodiment, the sample migration position can be determined byholding a pair of capillaries face to face with each other at aspecified gap, and two or more capillaries (the space between theadjacent capillaries set at 0.6 mm in the present embodiment) can belaid out at positions close to each other. This allows reduction in thesize of the optical cell. This also makes it possible to reduce thelaser light further to irradiate two or more gaps. Excitation lightdensity is increased, and fluorescence measurement is facilitated.

According to the present embodiment, buffer solution can be made to flowwithout using the mechanical means such as a liquid chromatography pump.This simplifies the apparatus configuration, and reduces the productioncost. There is no pulsating flow which may occur when the pump is used;this ensures a stable flow of sheath solution and reduced feed talevariation, and reduced variations of fluorescence intensity of samplesflowing in the gaps, resulting in detection accuracy. The flow rate ofsheath solution can be easily adjusted by changing the head between thesheath solution level in the sheath solution bottle and buffer solutionlevel in the vessel for an anode electrode on the downstream side formigration. This adjustment is also possible by changing the innerdiameter of the capillary on the downstream side for migration. It ispossible to make the sheath solution flow by using a mechanical meanssuch as a liquid chromatography pump. In this case, the advantage isthat the flow rate can be set directly. However, fluorescent intensitytends to change due to pulsating flow of the pump, so such treatment assmoothing in data processing is essential.

The sheath solution in the optical cell passes through capillaries 2a to2t on the downstream side and flows out of the cell. Since the capillarygenerally has a small inner diameter, the flow rate at the capillary isgenerally small. Therefore, the volume of the sheath solution can bereduced, resulting in improved maneuverability. In the presentembodiment, processing is made to ensure that the interior ofcapillaries 2a to 2t is positively charged, that the electroosmotic flowinside the capillaries 2a to 2t is directed toward the vessel for anodeelectrode, and that there is no reverse flow from capillaries 2a to 2tto the gaps. This ensures a stable flow of the sheath solution to vesselfor anode electrode even when the flow rate of the sheath solution issmall. If the inner diameter of capillaries 2a to 2t is great and theflow rate of the sheath solution in the gap is increased, the effect ofthe electroosmotic flow is reduced; as a result, treatment of the innersides of capillaries 2a to 2t is not necessary.

In the present embodiment, sheath solution and buffer solution in thevessel for a cathode electrode and the vessel for an anode electrode usethe same components as that of the buffer solution of the capillary gel.This prevents the capillary gel components from leaking into opticalcell 104a or the electrode vessel, and permits reuse of the capillarygel, resulting in stable electrophoresis featuring high separativepower. It is also possible to use the buffer solution which does notcontain urea, namely, DNA denaturant, but urea inside the capillary gelmay flow out from the capillary gel into the electrode vessel andfluorescent cell with the lapse of time. In this case, electrophoresisis possible as in the case of the buffer solution containing urea;however, the frequency of repeated use will reduce to some extent.

The used laser light source and fluorophores are not restricted to He-Nelaser and Texas Red (Sulforhodamine 101); any fluorophores and anysuitable laser light source can be used. The present embodiment has beendescribed based on the detection of the DNA fragments, but the principleapplies to the analysis of the protein and similar substances. In thepresent embodiment, two capillaries having the same inner diameters havebeen used; a combination of different inner diameters is also possible.For example, if the inner diameter of the capillary on the downstreamside is made smaller than that on the upstream side, the concentrationof the sample solution will be increased when samples migrating from theend of the upstream capillary are lead into the downstream capillary,resulting in increased sample concentration, hence highly sensitivedetection.

If the inner diameter of the capillary on the downstream side is madelarger than that on the upstream side, samples migrating from theupstream capillary can be lead into the downstream capillary withgreater ease and reliability.

Moreover, since fluorescence from the sample can be detected without theexcitation light passing through the capillary region according thepresent embodiment, the capillary need not be transparent. The capillarycoating need not be removed; this ensures handling ease. Furthermore, italso allows use of the capillary made of opaque fluorine-containedpolymer such as tetrafluoroethylene polymer tube and tri-fluoro ethylenechloride polymer.

The capillary made of fluorine-contained polymer features little suctionof samples, eliminating the need of treatment such as surface treatment.It is also very resistant against damage and chemicals, so use offluorine-contained polymer capillaries provides excellentmaneuverability, and permits use of solvents over an extensive range ofpH values. In the present embodiment, the case of two or more pairs ofcapillaries has been explained. In the case of one pair of capillaries,electrophoresis separation of samples, detection of the fluorescence anddetection of sample separation pattern are possible in the same way. Inthis case, the photomultiplier can be used as the optical detector.

EMBODIMENT 3

The following describes the DNA sequence determination method using theapparatus introduced in Embodiment 2. DNA fragments labeled byfluorophores are prepared by DNA polymerase reaction, using the primerlabeled by fluorophore according to the well-known dideoxy sequencingmethod invented by Sanger and his colleagues. The primer bonded withTexas Red (Sulforhodamine 101, maximum emission wavelength of 615 nm) isused as primer. Firstly, the labeled primer is added to thesingle-strand DNA and annealed, so that labeled primer is bonded to thesingle-strand DNA. This reaction solution is divided into four parts,which are subjected to DNA polymerase reactions corresponding to A, C, Gand T, respectively. That is, four types of deoxynucleotidetriphosphates (dATP, dTTP, dCTP and dGTP) and ddATP which will be aterminator are added to the single-strand DNA bonded with labeled primerto cause DNA polymerase reaction. The above procedure provides DNAfragments labeled by fluorophores having various lengths with terminalA. Similar reactions are made for C, G and T. Four types of reactionsolution obtained in the above procedure are poured into four of thecapillaries 1a to 1t, for example, capillaries 1a, 1b, 1c and 1d,respectively. The pouring procedure is the same as given in Embodiment2. Reaction solution A is put into capillary 1a, reaction solution Cinto 1b, reaction solution G into 1c and reaction solution T into 1d.After that, about 6 kV-voltage is applied to cause electrophoresis.Using the He-Ne laser having a wavelength of 594 nm to excite Texas Red(Sulforhodamine 101), change of the intensity of fluorescence in gaps 3ato 3d is measured with respect to time. Since the DNA fragments havingsmaller molecular weight are made to migrate earlier, the base sequenceis determined by analyzing the fluorescence intensity at each gapaccording to time sequence. The apparatus shown in Embodiment 2 has 20capillaries in which samples can be poured. Said DNA base sequencedetermination method allows simultaneous determination of base sequenceof five types of DNA samples. This permits determination of the basesequence of more DNA samples by increasing the number of pairs ofcapillaries held in the optical cell.

In the present embodiment, the Texas Red (Sulforhodamine 101) is used asfluorophore. However, it is also possible to detect fluorescence emittedfrom two or more fluorophores. In that case, for example, it is possibleto lake either of the following steps:

(1) the optical detector sets comprising interference filler 32, lens 33and detector 34 are prepared in numbers corresponding to that of thefluorophores, and each of the said detector sets is made to detectfluorescences of separate wavelength bands as shown in FIG. 4, or

(2) the splitting prism is installed after lens 33 to separate lightinto spectral components, and the image of each component is formed onthe two-dimensional detector such as a camera. Then fluorescenceintensity is measured for each migration lane and wavelength.

Thus, the DNA base sequence can be determined even when the apparatuswhich provides simultaneous detection of two or more fluorophores hasbeen configured. That is, each DNA is subjected to polymerase reaction,using primers labeled by different fluorophores for each type of theterminal base. Then reaction solutions are mixed and electrophoresis iscarried out; then fluorescence of the DNA fragments passing through thegap spaces is detected. The base type can be identified by identifyingthe fluorophore types, whereby the base sequence is determined.

The fluorophore types can be identified by comparing the fluorescencesat the maximum emission wavelength of four types of fluorophores. Inthis case, the base sequence of the DNA samples can be determined in themigration lane comprising a pair of capillaries and gaps. When two ormore migration lanes are present as shown in FIG. 3, the base sequenceof two or more DNA samples can be determined simultaneously in thenumber corresponding to the number of the migration lanes.

EMBODIMENT 4

The gap is formed by a pair of capillaries in said Embodiments 2 and 3,but the gap can also be made by other than the capillary pair. Forexample, the molecular weight separation region is composed ofcapillaries, as in said Embodiments. A gap can be formed by the end ofthese capillaries and the plate provided with fine holes. FIG. 5 showsthe configuration of the electrophoresis region and laser irradiationsystem of the electrophoresis apparatus according to the fourthEmbodiment. As in the case of Embodiment 2, twenty capillaries 1a to 1tas electrophoresis separation region are held by the multi-capillaryholder 5a and are fixed to the optical cell 60. The plate 62 providedwith fine holes 61a to 61t having an inner diameter of 200 μm is fixedon the side opposite to the optical cell 60. Fine holes 61a to 61t areprovided at the positions respectively corresponding to capillaries 1ato 1t, resulting in formation of the gaps. When the sheath solutionflows, the sample migrating from each capillary is led to eachcorresponding fine hole. The vessel for trapping solution 63 is laid outbelow plate 62 to temporarily store the solution coming from the fineholes, and the solution is then led to the vessel for electrode 65through tube 64.

Electrophoresis is performed in the same way as in Embodiment 2, byapplying power to the vessel for electrode 65 and another vessel forelectrode (not illustrated). Irradiation of laser and detection byfluorescence are also performed as in the case of the Embodiment 2.

EMBODIMENT 5

In Embodiment 2, it is also possible to design a configuration where thesamples are migrated by the flow of sheath solution, using only theupstream capillaries, without using the downstream ones. FIG. 6 showsthe configuration of the electrophoresis region of the electrophoresisapparatus. As in the case of Embodiment 2, ends of capillaries 1a to 1tare arranged and held in the optical cell 104a.

The bottom of optical cell 104a is connected with the seal plate 70 andtetrafluoroethylene polymer tube 71, and the end of tetrafluoroethylenepolymer tube 71 is immersed in the vessel for anode electrode 107, topermit flow of sheath solution. Sheath solution is poured in opticalcell 104a in the same way as in the case of the Embodiment 2.

Electrophoresis is performed by DC voltage applied from DC high voltagepower supply 12 between the vessel for cathode electrode 106 and thevessel for anode electrode 107. Immediately below the capillaries,samples which migrate from capillaries do not contact these sampleswhich migrate through other capillaries; they migrate through theoptical cell 104a, finally going to the vessel for anode electrode 107.Samples are detected by irradiating the laser light to 500 μm downstreamof the ends of the capillaries in optical cell 104a, and by receivingfluorescence.

The photodetecting system is designed in the same configuration as inEmbodiment 2. The sheath flow formation method of the present Embodimentis basically different from that of Embodiment 2. In the presentEmbodiment, sheath solution flows in the entire optical cell in alaminar flow. As a result, the flow rate of the sheath solution differsslightly, depending on the position inside the optical cell, and thesample migration lane also lends to vary; such unstable conditionsoccur. In terms of sensitivity and simplicity in the apparatusconfiguration, however, almost the same results can be obtained as thosein Embodiment 2.

EMBODIMENT 6

In the apparatus described in Embodiment 2, it is also possible to formthe gap by using the plate provided with two or more grooves instead ofthe downstream capillaries. FIG. 7 is an oblique view of the plateprovided with two or more grooves. Said Figure represents the case wherefour grooves corresponding to four capillaries are provided. Grooves81a, 81b, 81c and 81d are formed on one side of the plate 80 conformingto the form inside the optical cell. The form of the groove is made tocorrespond to the size of the capillary for electrophoresis separation;for example, the groove is designed to have a width of 300 μm and adepth of 600 μm. The pitch distance between grooves is 1 mm. Note thatthe pitch and groove form can be changed as required. FIG. 8 is anoblique view showing the sectional area of the optical cell ofelectrophoresis region of the electrophoresis apparatus using the saidplate. Optical cell 84 has inner dimensions of 20 mm in width, 3 mm indepth and 40 mm in length. The flow cell is used, which has the quartzglass plate having a thickness of 2 mm, with the top and bottom endsopened. Said Figure also shows the optical cell without the glass regionlocated in its front. Plate 80 is inserted into optical cell 84 in closecontact, so that the four lanes comprising the grooves 81a, 81b, 81c and81d, and the glass (not illustrated) in front of the optical cell areformed. Capillaries 82a, 82b, 82c and 82d having an inner diameter of100 μm and an outer diameter of 200 μm (gel formed inside in the sameprocedure as in Embodiment 2) are fixed inside the fluorescent cell 84by multi-capillary holder 83. The pitch distance between the capillariesare 1 mm so as to match the pitch distance between grooves, andadjustment is made to ensure that the capillary axis is located at thecenter of each groove. This adjustment is achieved by adjusting theposition of the holes on multi-capillary holder 83. Ends of capillaries82a, 82b, 82c and 82d are arranged inside the multi-capillary holder 83,and adjustment is made so that each capillary end will be about 1 mmaway from plate 80; then capillaries are fixed in the optical cell.

Electrophoresis is made possible by bringing the bottom of the opticalcell 84 in contact with the buffer solution inside the vessel for theanode electrode. As in the case of Embodiment 4 or 5, it is alsopossible to connect it to the vessel for the electrode through the tube.Sheath solution is poured in the same way as in the case of theEmbodiment 2. Samples migrating from the capillaries 82a to 82d are heldin the sheath solution and made to flow into optical cell 84; they arethen led to the vessel for the anode electrode through grooves 81a, 81b,81c and 81d.

Electrophoresis, laser irradiation and fluorescence detection areperformed in the same method as in the case of the Embodiment 2; themethod provides an effective and simultaneous detection of samplesseparated respectively by two or more capillaries. In the presentEmbodiment, the downstream side is formed by the plate constituting thegrooves, not by the capillaries. This allows easy adjustment of thedistance between each capillary end and the plate end ends of thegrooves. The plate is preferred to be made of quartz glass, wherefluorescence is not caused by laser irradiation.

EMBODIMENT 7

In the present Embodiment, the DNA fragments labeled by fluorophores areseparated by electrophoresis and are detected by fluorescence.Fluorescence detection is made at the region where the samples separatedby electrophoresis in the capillary migrate from the capillary end intothe buffer solution. The following describes the case of using the TexasRed (sulforhodamine 101) for labeling fluorophores:

FIG. 9 illustrates the configuration of the electrophoresis apparatus ofthe present Embodiment. It uses two capillaries; a silica capillary 101having an inner diameter of 100 μm, outer diameter of 375 μm and lengthof 30 cm and a silica capillary 102 having the same inner and outerdiameters with a length of 5 cm. Five percent polyacrylamide gelcontaining urea as denaturant is produced in capillaries 101 and 102, asin the case of the capillaries 1a and 1b in Embodiment 2.

Each one end of capillaries 101 and 102 is held face to face with theother in the optical cell, and the samples are detected using detectionof fluorescence. That is, each one end of capillaries 101 and 102 isheld face to face coaxially with the other forming a 0.1 nm gap 103inside rectangular quartz optical cell 104b (outer 3 mm square, inner 1mm square), and the gap space is used as an optical detecting portion.The other ends of capillary 101 and capillary 102 are immersedrespectively in the vessel for cathode electrode 106 and the vessel foranode electrode 107 filled with buffer solution (TRIS-borate-EDTA buffersolution). Buffer solution containing glycerin is poured into opticalcell 104b, and gap 103 is filled with buffer solution. DC high voltageis applied between the vessel for cathode electrode 106 and the vesselfor anode electrode 107 by DC high voltage power supply 12. When thevoltage is applied, current runs through capillary 102, gap 103 andcapillary 101. Gap 103 is short and, in gap 103, current flows along theline connecting the axes of capillary 102 and capillary 101. So themigrating sample flows out of capillary 101, and passes through gap 103without much dispersion, flowing into capillary 102. That is, the samplemigrates without contacting the optical cell 104b.

Buffer solution containing glycerin has been poured into optical cell104, namely, gap 103; addition of glycerin is intended to reduce theinfluence of convection due to increased viscosity of buffer solutionand to improve the electric field intensity in gap 103. Controlledconvection of the buffer solution and increased electric field intensityreduces the dispersion of the sample in gap 103, allowing easy migrationof the sample from capillary 101 to capillary 102.

Glycerin is used in the present Embodiment. Other substances can be usedif they have high viscosity and are usable for electrophoresis. Forexample, polyethylene glycol or succrose etc. can be used. If gap 103 issufficiently narrow, normal buffer solution without glycerin may beused. To introduce DNA fragments labeled by fluorophores which aresamples, one end of capillary 101 on the cathode side is immersed in thesample solution temporarily, and 5 kV voltage is applied between thesample solution and the vessel for anode electrode 107 for about 20seconds. Then the end of capillary 101 is returned to the vessel forcathode electrode 106, and 10 kV DC voltage is applied between thevessel for cathode electrode 106 and the vessel for anode electrode 107.Then the samples migrate from cathode to anode sides in capillary 101while undergoing molecular weight separation, and pass through gap 103.

Laser light 21 having a wavelength of 594 nm from the He-Ne laser source20 is condensed to about 20 μm by lens 22, and is irradiated on the gap103. Fluorescence 30 issued from the DNA fragments is collected by lens112 from the direction perpendicular to the direction in which laserlight is irradiated. Backgrounds such as scattered light are eliminatedby interference filler 113, and the image is formed on slit 115 by lens114. The light passing through slit 115 is detected by photomultiplier116, and amplified by amplifier 117; then the electrophoresis pattern isprocessed by data processor 118 of the computer or the like, and theirresults are displayed on monitor 37, and are output on printer 38 orstored in memory 39.

To ensure effective detection of the fluorescence issued from the TexasRead, interference filler 113 uses the band pass interference fillerwhich permits transmission of a wavelength band ranging from 610 to 630nm. Lens systems 112 and 114 are configured to have the equalmagnifications.

The opening of slit 115 is set to 50 μm in the direction ofelectrophoresis and 100 μm in the direction of laser light, andadjustment is made to ensure that fluorescent image in gap 3 is locatedat the center of the opening. Since samples migrate only close to gap 3,the image of the scattered light of the laser light at optical cell 104bis not formed on the opening of slit 115 and is not detected by thephotomultiplier 116 because of the configuration of said optical system.Scattered light and fluorescence are not produced by the capillary andgel, resulting in substantially reduced background intensity.

FIG. 10 is an enlarged view representing the sectional area of theoptical cell 104b. Quartz optical cell 104b is fixed by multi-capillaryholders 123a and 123b. Liquid leakage is prevented by insertion of thesilicone rubber packings 122a and 122b between optical cell 104b andholders 123a and 123b. Capillary 101 is fixed to multi-capillary holders123a by tetrafluoroethylene polymer ferrule 124a and screw 125a.Likewise, capillary 102 is also fixed by ferrule 124b and screw 125b.The gap between capillary 101 and capillary 102 can be freely adjustedby specifying the length of the capillary from the ferrule. In thepresent Embodiment, this is 0.1 mm, as described previously. The opticalcell is provided with solution inlet to pour buffer solution. It is alsoconnected with the valves 126a and 126b. The buffer outlet can beprovided at the optical cell holder. Optical cell 104b is filled withbuffer solution after fixing the capillary.

DNA fragments labeled by fluorophores can be dejected by the apparatusin the present Embodiment. The DNA fragments labeled by fluorophoresarea prepared according to the well-known dideoxy sequencing methodinvented by Sanger and his colleagues, under the same conditions as inthe case of the Embodiment. As discussed above, the DNA fragmentelectrophoresis pattern can be measured by introducing the samples intothe capillary and measuring the fluorescence intensity in the gap. As inthe case of the Embodiment 2, compared to the case of fluorescencedejection by irradiating the laser light on the capillary withoutcoating, the background intensity is reduced by about one tenth or lessin the present Embodiment, permitting dejection of samples of lessconcentration. The method according to the present Embodiment provideshighly sensitive detection of samples using the gel, without loss ofelectrophoresis characteristics. The buffer solution is not made toflow, so a simple apparatus configuration can be obtained. The laserequipment and fluorophores to be used are not restricted to He-Ne laserand Texas Red (sulforhodamine 101); any fluorophores and any suitablelaser light source can be used. Furthermore, as in the case of theEmbodiment 3, the present method provides simultaneous fluorescencedetection of two or more fluorophores. When the DNA base sequence is tobe determined using the two or more fluorophores in the apparatusaccording to the present Embodiment, exactly the same procedure as inthe case of the Embodiment 3 is used. The present Embodiment has beenexplained with reference to the detection of DNA fragments, but the samemethod is applicable also to the analysis of protein, sugar and similarsubstances. Furthermore, two capillaries having the same inner diameterhave been used for the description of the present Embodiment. It is alsopossible to use a combination of capillaries having different innerdiameters. The effect resulting from the difference between the innerdiameters of capillaries 101 and 102 is the same as that described withreference to Embodiment 2. Furthermore, the method for the presentEmbodiment permits detection of the fluorescence without the excitationlight passing through the capillary region, so the capillary need not betransparent; the capillary coating need not be removed. It allows use ofcapillaries made of an opaque fluorine-contained polymer such astetrafluoroethylene polymer or trifluoro ethylene chloride polymer.

EMBODIMENT 8

The following describes the simultaneous fluorescence detectionapparatus with four capillaries arranged in parallel. The number ofcapillaries is not limited to four; it can be any number. FIG. 11 showsthe configuration of the electrophoresis apparatus for the presentEmbodiment. The 127a, 127b, 127c and 127d in the Figure denote silicacapillaries having an inner diameter of 100 μm, outer diameter of 375 μmand length of 30 cm. One end of each of the capillaries (not shown) isimmersed in the vessel for the cathode electrode as in the case shown inFIG. 9. Likewise, 128a, 128b, 128c and 128d denote the silicacapillaries having the same inner and outer diameters with a length of 5cm, and one end of each is immersed in the vessel for the anodeelectrode (not shown). According to the same method as given in theEmbodiment 2, five percent polyacrylamide gel containing urea asdenaturant is produced in these capillaries.

Each capillary is provided with silane coupling treatment so thatpolyacrylamide gel and capillary wall are chemically bonded together,with consideration given so as not to allow the capillary to elude fromthe acrylamide during electrophoresis.

Capillary 127a to 127d and capillaries 128a to 128d are held and fixedin rectangular quartz optical cell 104c, maintaining gaps 129a to 129dof specified intervals, as in the case of the Embodiment 7, and arearranged in a straight line in close proximity with each other, formingtwo or more gaps, namely, optical detecting portions.

The capillaries are fixed by the capillary holders 142 and 143 of theblock made of fluorine-contained polymer such as tetrafluoroethylene,provided with four vertical holes at intervals of 1 mm. Each capillaryis inserted into each of four vertical holes, and adjustment is so madethat 127a and 128a, 127b and 128b, 127c and 128c, and 127d and 128d arerespectively coaxial, and that each length of gaps 129a to 129d will be0.2 mm.

Buffer solution containing glycerin is poured into the optical cell104c, filling gaps 129a to 129d with buffer solution. If voltage isapplied under this condition, the sample led into capillary 127amigrates to gap 129a, then to 128a. Samples led to other capillariesalso migrate in the same way. The glycerin in the buffer solutionrestrains the convection of the buffer solution in gaps, as in the caseof embodiment 7 and ensures reliable and smooth migration of the samplefrom capillaries 127 a to 127d to capillaries 128a to 128d.

Fluorescent detection of the DNA fragments passing through gaps 129a to129d is carried out as follows: Firstly, laser light 21 from lasersource 20 is condensed by lens 22 to permit simultaneous irradiation oftwo or more optical detecting portions, namely, gaps 129a to 129d. Thefocal point is set at the mid-position between the gaps 129b and 129c,and adjustment is made to ensure that the maximum diameter of theirradiated spot size is 100 μm between gaps 129a to 129d. When the focaldistance of about 150 mm is used as lens 22, for example, it is possibleto irradiate gaps 129a to 129d with much the same spot size.

Fluorescence 30 emitted from DNA fragments passing through each gap iscollected by lens 134 from the position perpendicular to the directionin which the laser is irradiated, and the background such as scatteredlight is removed by the interference filter 32. Then it is collected bylens 33, and the image is formed on line sensor 37 such as a photodiodearray or CCD camera. In the configuration in FIG. 11, using thefluorescence detection system as in the case of the Embodiment 1 shownin FIG. 1, it is also possible to collect the fluorescence from thelinear laser irradiation region by the cylindrical lens. After the datais output from the output of line sensor 37, the electrophoresis patternand the like is subjected to data processing by data processor 36 of thecomputer or similar device, and their results are displayed on monitor37, and are output on printer 38 or stored in memory 39.

Using the He-Ne laser having a wavelength of 594 nm as a laser light andTexas Red (sulforhodamine 101) solution as sample, the sample is made tomigrate in capillaries 127a to 127d. When fluorescent images of samplesmigrating in gap spaces which are optical detectors are detected by theline sensor 37, strong fluorescence intensity is detected in fourpositions of the gaps 129a to 129d, namely, optical detecting portionscorresponding to migration lanes, each independently of the otheroptical detecting portions. Continuous and simultaneous detection of thesamples migrating in the capillaries is possible by detecting theintensity of the signals at the positions corresponding to migrationlanes on the line sensor.

The method according to the present Embodiment allows fluorescencedetection of samples migrating simultaneously and under the sameconditions by two or more optical detectors. It also allows analysisover an extensive range, for example, DNA base sequence determinationfor one or more samples, analysis of the side strand or functional groupusing the fluorophores, and detailed analysis of the samples separatedby liquid chromatography. Furthermore, pairs of capillaries are arrangedin a straight line in close proximity to form two or more gaps, so onlyone fluorescence collecting means such as a lens is sufficient; thisfeature ensures reduced cost and simple structure.

Furthermore, as in the case of the preceding Embodiments, laser light isirradiated along the gaps where there is no capillary. Then the laser isnot scattered or refracted by the capillary, so each gap can beirradiated by much the same spot size, with much he same intensity andreduced background intensity. This permits simple configuration of theapparatus which provides effective excitation of two or more samples,hence highly sensitive detection. Such an apparatus facilitatessimultaneous detection of many samples.

The DNA base sequence determination method using the apparatus accordingto the method of the present Embodiment is the same as that ofEmbodiment 3. Use of the Texas Red (sulforhodamine 101, maximum emissionwavelength: 615 nm) as a labeling fluorophore, and the He-Ne laserhaving a wavelength of 594 nm as an excitation light source isespecially preferred to ensure high sensitivity.

EMBODIMENT 9

The gap is formed by a pair of capillaries in said Embodiments 7 to 8,but it can also be by other substances than capillaries. For example,the electrophoresis separation region is composed of capillaries, as insaid Embodiments. A gap can be formed by the end of these capillariesand the plate provided with fine holes. FIG. 12 is an enlarged viewrepresenting the sectional area of the optical cell in which the gap isformed by a capillary and the plate provided with fine holes.

As in the case of the Embodiment 7, capillary 171 filled withpolyacrylamide gel and plate 173 provided with fine holes 172 (forexample, a plate made of tetrafluoroethylene polymer) are arranged faceto face so that the axis of capillary 171 and that of fine hole 172 willbe almost matched to each other, and gap 174 is formed. They are heldinside the optical cell 175. This cell is a rectangular optical cellhaving outer dimensions of 3 mm square and inner dimensions of 1 mmsquare, with the top and bottom ends opened. It is held, for example, bytetrafluoroethylene polymer block 176 on the top, and held by plate 173on the bottom and fixed, so that buffer solution can be stored in thecell. The tetrafluoroethylene polymer block 176 is provided with thehole conforming to the outer dimensions of capillary 171. Capillary 171is passed through that hole to hold the capillary 171, and the length ofgap 174 can be adjusted from 0.1 mm to 1.0 mm. The other end ofcapillary 171 is immersed in the vessel for the cathode electrode, as inthe case of the Embodiment 7. The tetrafluoroethylene polymer tube 177is connected to the bottom of plate 173 through tube holder 178, and isled to the vessel for the anode electrode. Plate 173 can also be made tocontact with the vessel for the anode electrode. Although not shown inFIG. 12, the buffer solution inlet is provided inside fluorescent cell175, as in the case of FIG. 10. The buffer solution is supplied throughthe valve during electrophoresis, to fill optical cell 175, gap 174 andtetrafluoroethylene polymer tube 177. After the supply of buffersolution is slopped, the sample is made to migrate. The method of thepresent Embodiment provides fluorescent detection with the minimumbackground intensity as in the case of Embodiment 7, and highlysensitive fluorescence detection.

Arrangement of fine holes on plate 173 in a straight line permitsconfiguration of the optical cell which has the same effects as those inthe case of the Embodiment 8.

The method of the present Embodiment ensures easy installation of theapparatus since plate 173 is used on one side.

As disclosed above, according to the present invention, one or moresamples are separated by electrophoresis separation by using the plategel or capillary gel, and the laser beam is irradiated linearly on thetwo or more migration lanes from direction which is approximatelyperpendicular to the direction for sample migration and which isparallel the surface formed by two or more migration lanes; therebydetecting fluorescence on a real-time basis from the fragments migratingtwo or more migration lanes. Use of Texas Red (sulforhodamine 101,maximum emission wavelength: 615 nm) as labeling fluorophore, and He-Nelaser light having an emission wavelength of 594 nm as an excitationlight source is particularly preferred to ensure high sensitivity inEmbodiments 1 to 9. In Embodiments 2 to 9, however, the fluorophore andlight source to be used are not restricted to Texas Red and He-Ne laserhaving an emission length of 594 nm. For example, rhodamine derivatives,FITC, SF, TRITC and ALPC (aluminum phthalocyanine) complex (emissionwavelength: about 700 nm) as fluorophore, and argon ion laser (emissionwavelength: 488 nm and 514.5 nm), He-Ne laser (emission wavelength:632.8 nm), YAG laser (second harmonic wavelength: 532 nm) andsemiconductor laser (emission wavelength: about 670 nm) as the laserlight source can be used.

What is claimed is:
 1. An electrophoresis apparatus comprising:aplurality of first capillaries each containing a separation medium, theseparation medium having samples labeled with fluorophores disposedtherein; a plurality of second capillaries each containing a buffersolution; an optical cell filled with the buffer solution and having oneend of each of the first capillaries and one end of each of the secondcapillaries disposed therein with the ends of the second capillariesbeing separated from respective ones of the ends of the firstcapillaries by respective gaps; sheath flow forming means forintroducing the buffer solution into the optical cell and formingrespective sheath flows of the buffer solution around the gaps; meansfor generating an electric field for causing the samples labeled withthe fluorophores to migrate through the first capillaries into the gaps;a light source for exciting the fluorophores with light when the sampleslabeled with the fluorophores are in the gaps, thereby causing thefluorophores to emit fluorescence in the gaps; and photodetecting meansfor detecting the fluorescence emitted by the fluorophores in the gaps.2. An electrophoresis apparatus according to claim 1, wherein theseparation medium is a gel.
 3. An electrophoresis apparatus according toclaim 1, wherein each of the gaps is between 0.1 mm and 3 mm.
 4. Anelectrophoresis apparatus according to claim 1, wherein the gaps arearranged in a straight line; andwherein the light source excites thefluorophores in the gaps substantially simultaneously.
 5. Anelectrophoresis apparatus according to claim 1, wherein the light sourceincludes a He-Ne laser emitting light having a wavelength of 594 nm forexciting the fluorophores.
 6. An electrophoresis apparatus according toclaim 1, wherein the fluorophores include either sulforhodamine or aderivative of sulforhodamine.
 7. An electrophoresis apparatuscomprising:a plurality of capillaries each containing a separationmedium, the separation medium having samples labeled with fluorophoresdisposed therein; an optical cell having one end of each of thecapillaries disposed therein; sheath flow forming means for introducinga sheath solution into the optical cell and forming respective sheathflows of the sheath solution around the ends of the capillaries; meansfor generating an electric field for causing the samples labeled withthe fluorophores to migrate through the capillaries into the sheathflows; a light source for exciting the fluorophores with light when thesamples labeled with the fluorophores are in the sheath flows, therebycausing the fluorophores to emit fluorescence in the sheath flows; andphotodetecting means for detecting the fluorescence emitted by thefluorophores in the sheath flows.
 8. An electrophoresis apparatusaccording to claim 7, wherein the separation medium is a gel.
 9. Anelectrophoresis apparatus according to claim 7, wherein the ends of thecapillaries are arranged in a straight line;wherein the light sourceexcites the fluorophores in the sheath flows substantiallysimultaneously; and wherein the photodetecting means detects thefluorescence emitted by the fluorophores in the sheath flowssubstantially simultaneously.
 10. An electrophoresis apparatus accordingto claim 7, wherein the light source includes a He-Ne laser emittinglight having a wavelength of 594 nm for exciting the fluorophores. 11.An electrophoresis apparatus according to claim 7, wherein thefluorophores are either sulforhodamine or a derivative ofsulforhodamine.
 12. An electrophoresis apparatus comprising:a pluralityof first capillaries each containing a separation medium, the separationmedium having samples disposed therein; an optical cell filled with abuffer solution and having one end of each of the first capillariesdisposed therein; means for generating an electric field for causing thesamples to migrate through the first capillaries into the buffersolution; and optical detecting means for detecting the samples when thesamples are in the buffer solution.
 13. An electrophoresis apparatusaccording to claim 12, wherein the separation medium is a gel.
 14. Anelectrophoresis apparatus according to claim 12, further comprisingsheath flow forming means for introducing the buffer solution into theoptical cell and forming respective sheath flows of the buffer solutionaround the ends of the first capillaries to prevent the samples frombeing diffused in the buffer solution when the samples migrate into thebuffer solution.
 15. An electrophoresis apparatus according to claim 12,further comprising a plurality of second capillaries;wherein one end ofeach of the second capillaries is disposed in the optical cell such thatthe ends of the second capillaries are separated from the ends of thefirst capillaries by respective gaps; wherein the electric field causesthe samples to migrate through the first capillaries into the secondcapillaries through the gaps; and wherein the buffer solution flows outof the optical cell through the second capillaries.
 16. Anelectrophoresis apparatus according to claim 15, wherein each of thegaps is between 0.1 mm and 3 mm.
 17. An electrophoresis apparatusaccording to claim 15, wherein the gaps are arranged in a straight line;andwherein the optical detecting means detects the samples in the gapssubstantially simultaneously.
 18. An electrophoresis apparatus accordingto claim 12, further comprising means for causing the buffer solution toflow along an outside peripheral surface of each of the firstcapillaries so as to form respective sheath flows of the buffer solutionaround the first capillaries.
 19. An electrophoresis apparatus accordingto claim 12, further comprising a single second capillary;wherein oneend of the single second capillary is disposed in the optical cell suchthat the end of the single second capillary is separated from the endsof the first capillaries; wherein the electric field causes the samplesto migrate through the first capillaries into the single secondcapillary through the buffer solution; and wherein the buffer solutionflows out of the optical cell through the single second capillary. 20.An electrophoresis apparatus according to claim 12, wherein the ends ofthe first capillaries are arranged in a straight line; andwherein theoptical detecting means detects the samples when the samples are in thebuffer solution outside the ends of the first capillaries substantiallysimultaneously.
 21. An electrophoresis apparatus according to claim 12,wherein the samples are labeled with fluorophores; andwherein theoptical detecting means includes: a light source for exciting thefluorophores with light when the samples labeled with the fluorophoresare in the buffer solution, thereby causing the fluorophores to emitfluorescence in the buffer solution; and photodetecting means fordetecting the samples by detecting the fluorescence emitted by thefluorophores in the buffer solution.
 22. An electrophoresis apparatusaccording to claim 12, wherein the optical detecting means includes:alight source for irradiating the samples with light when the samples arein the buffer solution; and photodetecting means for detecting thesamples by detecting light absorption by the samples irradiated with thelight in the buffer solution.
 23. An electrophoresis apparatuscomprising:a plurality of first capillaries each containing a separationmedium, the separation medium having samples labeled with fluorophoresdisposed therein; a plurality of second capillaries each containing abuffer solution; an optical cell filled with the buffer solution andhaving one end of each of the first capillaries and one end of each ofthe second capillaries opening therein with respective gapstherebetween; sheath flow forming means for introducing the buffersolution into the optical cell and forming respective sheath flows ofthe buffer solution around the first capillaries; means for generatingan electric field for causing the samples labeled with the fluorophoresto migrate through the first capillaries into the gaps; a light sourcefor exciting the fluorophores with light when the samples labeled withthe fluorophores are in the gaps, thereby causing the fluorophores toemit fluorescence in the gaps; and photodetecting means for detectingthe fluorescence emitted by the fluorophores in the gaps.
 24. Anelectrophoresis apparatus according to claim 23, wherein the separationmedium is a gel.
 25. An electrophoresis apparatus according to claim 23,wherein each of the gaps is between 0.1 mm and 3 mm.
 26. Anelectrophoresis apparatus according to claim 23, wherein the gaps arearranged in a straight line; andwherein the light source excites thefluorophores in the gaps substantially simultaneously.
 27. Anelectrophoresis apparatus according to claim 23, wherein the lightsource includes a He-Ne laser emitting light having a wavelength of 594nm for exciting the fluorophores.
 28. An electrophoresis apparatuscomprising:a plurality of first capillaries each containing a separationmedium, the separation medium having samples labeled with fluorophoresdisposed therein; a single second capillary; an optical cell having oneend of each of the first capillaries and one end of the single secondcapillary disposed therein with the end of the single second capillarybeing separated from the ends of the first capillaries; sheath flowforming means for introducing a sheath solution into the optical celland forming respective sheath flows of the sheath solution around theends of the first capillaries, wherein the sheath solution flows out ofthe optical cell through the single second capillary; means forgenerating an electric field for causing the samples labeled with thefluorophores to migrate through the first capillaries into the sheathflows; a light source for exciting the fluorophores with light when thesamples labeled with the fluorophores are in the sheath flows, therebycausing the fluorophores to emit fluorescence in the sheath flows; andphotodetecting means for detecting the fluorescence emitted by thefluorophores in the sheath flows.
 29. An electrophoresis apparatusaccording to claim 28, wherein the separation medium is a gel.
 30. Anelectrophoresis apparatus according to claim 28, wherein the ends of thefirst capillaries are arranged in a straight line;wherein the lightsource excites the fluorophores in the sheath flows substantiallysimultaneously; and wherein the photodetecting means detects thefluorescence emitted by the fluorophores in the sheath flowssubstantially simultaneously.
 31. An electrophoresis apparatuscomprising:a plurality of capillaries each containing a separationmedium, the separation medium having samples disposed therein; anoptical cell having one end of each of the capillaries disposed therein;sheath flow forming means for forming respective sheath flows of asheath solution at the ends of the capillaries disposed in the opticalcell; means for generating an electric field for causing the samples tomigrate through the capillaries into the sheath flows; and opticaldetecting means for detecting the samples when the samples are in thesheath flows.
 32. An electrophoresis apparatus comprising:a plurality ofcapillaries each containing a separation medium, the separation mediumhaving samples disposed therein; an optical cell having one end of eachof the capillaries disposed therein; a vessel filled with a sheathsolution, the vessel being disposed outside the optical cell and beingconnected to the optical cell such that the sheath solution flows intothe optical cell and forms respective sheath flows of the sheathsolution at the ends of the capillaries; means for generating anelectric field for causing the samples to migrate through thecapillaries into the sheath flows; a light source for irradiating thesamples with light when the samples are in the sheath flows; and opticaldetecting means for detecting the samples irradiated with the light inthe sheath flows.
 33. An electrophoresis apparatus comprising:aplurality of capillaries each containing a separation medium, theseparation medium having samples disposed therein; an optical cellhaving one end of each of the capillaries disposed therein; sheath flowforming means for causing a sheath solution to flow along an outsideperipheral surface of each of the capillaries to form respective sheathflows of the sheath solution around the ends of the capillaries disposedin the optical cell; means for generating an electric field for causingthe samples to migrate through the capillaries into the sheath flows;and optical detecting means for detecting the samples when the samplesare in the sheath flows.
 34. An electrophoresis apparatus comprising:afirst capillary having samples disposed therein; a second capillary; anoptical cell having one end of the first capillary and one end of thesecond capillary disposed therein with the end of the second capillarybeing separated from the end of the first capillary by a gap; sheathflow forming means for forming a sheath flow of a sheath solution in theoptical cell around the gap; means for generating an electric field forcausing the samples to migrate through the first capillary into the gapwithin the sheath flow; and optical detecting means for detecting thesamples when the samples are in the gap within the sheath flow.
 35. Anelectrophoresis apparatus comprising:a plurality of capillaries eachhaving samples disposed therein; an optical cell having one end of eachof the capillaries disposed therein; sheath flow forming means forforming respective sheath flows of a sheath solution in the optical cellat the ends of the capillaries; means for generating an electric fieldfor causing the samples to migrate through the capillaries into thesheath flows; and optical detecting means for detecting the samples whenthe samples are in the sheath flows.
 36. An electrophoresis apparatuscomprising:a plurality of capillaries each containing a separationmedium, the separation medium having samples disposed therein; anoptical cell filled with a buffer solution and having one end of each ofthe capillaries disposed therein; means for generating an electric fieldfor causing the samples to migrate through the capillaries into thebuffer solution; a light source for substantially simultaneouslyirradiating the samples with light when the samples are in the buffersolution outside the ends of the capillaries; and optical detectingmeans for substantially simultaneously detecting the samples when thesamples are in the buffer solution outside the ends of the capillaries.37. An electrophoresis apparatus comprising:a plurality of capillarieseach containing a separation medium, the separation medium havingsamples disposed therein; sheath flow forming means for forming a sheathflow of a sheath solution at one end of each of the capillaries; meansfor generating an electric field for causing the samples to migratethrough the capillaries into the sheath flows; a light source forsubstantially simultaneously irradiating the samples with light when thesamples are in the sheath flows, the light passing through the sheathflows continuously; and optical detecting means for substantiallysimultaneously detecting the samples when the samples are in the sheathflows.
 38. An electrophoresis apparatus comprising:a plurality ofcapillaries each containing a separation medium, the separation mediumhaving samples disposed therein; sheath flow forming means for causing asheath solution to flow along an outside peripheral surface of each ofthe capillaries to form respective sheath flows of the sheath solutionaround the capillaries; means for generating an electric field forcausing the samples to migrate through the capillaries into the sheathflows; a light source for substantially simultaneously irradiating thesamples with light when the samples are in the sheath flows, the lightpassing through the sheath flows continuously; and optical detectingmeans for substantially simultaneously detecting the samples when thesamples are in the sheath flows.
 39. An electrophoresis apparatuscomprising:a first capillary having samples disposed therein; a secondcapillary; an optical cell having one end of the first capillary and oneend of the second capillary disposed therein with the end of the secondcapillary being separated from the end of the first capillary by a gap;sheath flow forming means for forming a sheath flow of a sheath solutionin the optical cell around the gap; means for generating an electricfield for causing the samples to migrate through the first capillaryinto the gap within the sheath flow; a light source for substantiallysimultaneously irradiating the samples with light when the samples arein the gap within the sheath flow; and optical detecting means forsubstantially simultaneously detecting the samples when the samples arein the gap within the sheath flow.
 40. An electrophoresis apparatuscomprising:a plurality of capillaries each having samples disposedtherein; an optical cell having one end of each of the capillariesdisposed therein; sheath flow forming means for forming respectivesheath flows of a sheath solution in the optical cell at the ends of thecapillaries; means for generating an electric field for causing thesamples to migrate through the capillaries into the sheath flows; alight source for substantially simultaneously irradiating the sampleswith light when the samples are in the sheath flows; and opticaldetecting means for substantially simultaneously detecting the sampleswhen the samples are in the sheath flows.